The goal of the proposed research is to understand biological energy transduction between the cytoplasmic and outer membranes of Gram-negative bacteria, using Escherichia coli as the model system. in E. coli, cytoplasmic membrane energy is coupled to the active transport of important nutrients (iron-siderophore complexes and vitamin Bl2) across the outer membrane. Although we know that energy transduction is energized by the cytoplasmic membrane electrochemical potential, and that the proteins TonB, ExbB, and ExbD participate in that process, the molecular mechanism of energy transduction and the role of ExbB and ExbD are unclear. The role of TonB as energy transducer has been established, however, the molecular details of its interactions with other energy transduction proteins have not been elucidated. Furthermore, recent data suggest the participation of additional proteins which have been defined only biochemically, and not genetically. The aims of this proposal are to 1.) define, both genetically and biochemically, the protein components of the energy transduction machinery; 2.) determine the function(s) of specific amino acid residues in the amino terminus of the energy transducer, TonB. These are the regions of TonB believed to be in direct contact with the energy generating machinery in the cytoplasmic membrane. 3.) Investigate the role of ExbB in energy transduction, focusing on its proposed recycling activity and the function of its cytoplasmic domains.